STRUCTURAL ANALYSIS OF PROTEIN FOLDING INTERMEDIATES

蛋白质折叠中间体的结构分析

• 批准号: 2022887
• 负责人: Robert O. Fox
• 金额: $ 21.24万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022887
• 关键词: X ray crystallography; chemical cleavage; chemical kinetics; conformation; myoglobin; nuclear magnetic resonance spectroscopy; nuclease; protein folding; protein sequence; protein structure; structural biology

While may small proteins can refold spontaneously in vitro, the identification of the folding pathway, and the detection and structural characterization of folding intermediates have been difficult. Recently, attention has turned to the molten globule state and other nonnative equilibrium states of proteins which are thought to be models for kinetic intermediates in protein folding. Fragments of staphylococcal nuclease have been produced which also have properties similar to those of the molten globule, i.e. a somewhat compact structure with some secondary structure but without a defined tertiary structure. Pulsed hydrogen-deuterium exchange during refolding has been used to probe the protection of backbone amide hydrogens from solvent exchange during refolding of a number of proteins and most recently the staphylococcal nuclease Pro 117 yields Gly variant. The extent of exchange for 39 residues is determined by two-dimensional proton NMR after refolding for 5 ms to 10s. Three kinetic phases are inferred. Modest protection of amides in the early refolding intermediate composed to two beta-sheets formed by local sequence interactions is observed after a 5 ms refolding period. Native levels of protection throughout the molecule accrue more slowly in tow kinetic phases (k approximately 2s-1, k less than 0.01s-1). Protection factors were determined by varying the high pH labeling pulse after refolding for 100 ms. Little or no native or unfolded protein is present; instead, most molecules are in one or more partially folded states. The intermediate state has modest, yet significant, protection for residues in the beta-sheets (protection factors 10-60), and almost no protection in the alpha- helices (protection factors less than 10). The pattern of labeling is consistent with a role for beta turns and beta-hairpins in the formation of the early intermediate. Recently a chemical cleavage method has been developed where an EDT-Fe based reagent (EPD-Fe) can be attached to a protein via a cysteine side chain. The addition of ascorbate generates hydroxyl radicals at the iron center which diffuse and cleave the polypeptide backbone in a region close to the cysteine attachment site at residues accessible to solvent. The observed cleavage sites can be mapped by amino acid sequencing. The cleavage is dependent on protein conformation. We propose characterize the molten globule state of apomyoglobin and staphylococcal nuclease fragment structures using this newly developed chemical cleavage technique. We will prepare a number of cysteine variants of these proteins and characterize the cleavage patterns observed in the native and molten globule states.

虽然可能的小蛋白在体外可以自发折叠，但 折叠途径的鉴定、检测和结构 折叠中间体的表征一直很困难。 最近，人们的注意力转向了熔化的球状状态和其他 被认为是模型的蛋白质的非自然平衡状态 用于蛋白质折叠的动力学中间体。零碎的 产生了葡萄球菌核酸酶，它也具有 类似于熔化的球体，即有点致密 具有一些二级结构但没有定义的三级结构的结构 结构。 在复性过程中的脉冲氢-氚交换已被用于 保护主链酰胺氢不受溶剂交换的影响 在一些蛋白质的重折叠过程中，最近 葡萄球菌核酸酶Pro 117产生Gly变异体。其影响范围 用二维质子核磁共振确定39个残基的交换 再折叠5ms至10s后。推论出三个动力学阶段。 酰胺类化合物在合成的早期复性中间体中的适度保护 观察到由局部序列相互作用形成的两个β-折叠 在5毫秒的重折叠周期之后。自始至终的本机保护级别 分子在两个动力学阶段中增长较慢(约k 2s-1，k小于0.01s-1)。保护系数由以下因素确定 在复性100ms后改变高pH标记脉冲。一点儿 或者不存在天然的或未折叠的蛋白质；相反，大多数分子是 处于一个或多个部分折叠状态。中间状态具有 对Beta-Sheet中的残留物提供适度但重要的保护 (保护系数10-60)，而在阿尔法- 螺旋体(保护系数小于10)。标签的模式是 与贝塔转弯和贝塔发夹在形成中的作用一致 早期中期的。 最近开发了一种化学切割方法，其中EDT-Fe 碱性试剂(EPD-Fe)可以通过半胱氨酸侧连接到蛋白质上 链条。抗坏血酸的加入会在 铁中心，它扩散和切割多肽主干在一个 接近半胱氨酸结合部位的区域，其残基可达 溶剂型。观察到的裂解位点可以用氨基酸作图。 测序。这种切割依赖于蛋白质的构象。我们 建议表征去肌红蛋白的熔融球状状态和 利用这一新开发的葡萄球菌核酸酶片段结构 化学卵裂技术。我们会准备一些半胱氨酸 这些蛋白质的变异体，并表征切割模式 在天然状态和熔融状态下观察到的。